SUMMARY The long-term objective of this research program is to define the mechanisms by which the central hemostatic protease, thrombin, contributes to the development of sickle cell disease (SCD) pathologies. The seminal role of thrombin in hemostasis and vascular biology is underscored by the fact that this protease positively controls fibrin deposition, platelet activation, and endothelial cell (EC) signaling events via multiple substrates and receptors [e.g., protease-activated receptors, fibrinogen, factor XI, and factor XIII], as well as negatively controls further thrombin generation through the activation of protein C, a natural anticoagulant with known anti-inflammatory/cytoprotective properties. The control of thrombin activity has been intensely studied because thrombin-mediated proteolysis is fundamental to both physiological hemostasis and pathological vaso-occlusive events, including myocardial infarction, venous thrombosis and stroke. However, an additional driving force for detailed studies of thrombin and thrombin targets is that these proteins also control vascular permeability/barrier function, tissue repair, and inflammation, which together contribute to the development of multiple inflammatory diseases. Given that circulating sickle cells result in a combination of vascular damage, occlusive events and inflammatory changes, and given that local and systemic hemostatic system activation is a conspicuous feature of SCD, thrombin and thrombin targets are prime candidates to be clinically-significant modifiers of sickle cell disease pathobiology. The aims of this project center on two general hypotheses: i) thrombin, as a master regulator of vascular biology, platelet/EC activation, fibrin deposition and inflammatory processes, is a major determinant of SCD pathologies, and ii) SCD-associated morbidities can be ameliorated by novel genetic or pharmacological interventions at the level of pro/thrombin and downstream thrombin substrates. These hypotheses will be tested through studies that focus on defining the importance of prothrombin in the development of multi-organ SCD pathologies and long-term survival in Berkeley sickle mice (Hba0/Hbb0 [Tg(Human HbS)]+/+) (Aim 1); understanding the thrombin-mediated, fibrin(ogen)-dependent and fibrin(ogen)-independent mechanisms driving SCD pathologies (Aim 2); and establishing the potential benefit of thrombin-targeted pharmacological intervention in limiting SCD pathologies in mice (Aim 3). The proposed studies will provide for the first time a clear understanding of the significance of hemostatic factors in the pathogenesis of SCD and may illuminate novel therapeutic strategies for limiting SCD-induced morbidities.